Ligand Binding Strategies of Human Serum Albumin: How Can the Cargo Be Utilized?

Review ArticleLigand Binding Strategies of Human Serum Albumin:

How Can the Cargo be Utilized?ANKITA VARSHNEY,1 PRIYANKAR SEN,1 EJAZ AHMAD,1 MOHD. REHAN,2

NAIDU SUBBARAO,2 AND RIZWAN HASAN KHAN1*1Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India

2Centre for Computational Biology & Bioinformatics, School of Information Technology,Jawaharlal Nehru University, New Delhi, India

ABSTRACT Human serum albumin (HSA), being the most abundant carrier proteinin blood and a modern day clinical tool for drug delivery, attracts high attention amongbiologists. Hence, its unfolding/refolding strategies and exogenous/endogenous ligandbinding preference are of immense use in therapeutics and clinical biochemistry.Among its fellow proteins albumin is known to carry almost every small molecule.Thus, it is a potential contender for being a molecular cargo/or nanovehicle for clinical,biophysical and industrial purposes. Nonetheless, its structure and function are largelyregulated by various chemical and physical factors to accommodate HSA to its func-tional purpose. This multifunctional protein also possesses enzymatic properties whichmay be used to convert prodrugs to active therapeutics. This review aims to highlightcurrent overview on the binding strategies of protein to various ligands that may beexpected to lead to significant clinical applications. Chirality 00:000–000, 2009. VVC 2009

Wiley-Liss, Inc.

KEY WORDS: human serum albumin; ligand binding; enantioselectivity; clinicalapplications

INTRODUCTION

Human serum albumin (HSA) is an abundant, multifunc-tional and nonglycosylated, negatively charged plasmaprotein, with ascribed ligand-binding and transport proper-ties, antioxidant functions, and enzymatic activities.1 Theprotein is a monomer of 585 amino acid residue havingthree homologous a-helical domains, named domain I, do-main II and domain III.2 Each domain contains 10 helicesand is divided into antiparallel six-helix and four-helix sub-domains (A and B). Albumin is the most abundant serumprotein that serves as a transport vehicle for several en-dogenous compounds including fatty acids (FA), hemin,bilirubin, and tryptophan, all of which bind with high affin-ity.3,4 For a long time, albumin has attracted the attentionof the pharmaceutical industry because of its ability tobind a wide variety of drug molecules and alter their phar-macokinetic parameters.5 Although most ligands for albu-min are hydrophobic or anionic but heavy metals are alsoknown to bind to the protein.3–7 Very few cationic drugsare also known to bind with HSA. This versatility, whicharises from the presence of multiple binding sites, whichare dependent on various environmental factors like pH,temperature and ionic strength, makes it far from trivial toobtain detailed information on ligand binding. It has beenespecially difficult to assess the interactions between dif-ferent ligands for the protein even though it is a key for

our understanding of the role of HSA in vivo. Simultane-ous binding of several ligands to HSA molecule is a com-plex situation in which the protein recognizes individualligands by specific and nonspecific interactions. This rec-ognition is in turn strongly dependent on the microenvir-onment of the protein as it changes its conformationin vivo. In physiological condition the protein experiencedvarious environments like pH, varying ionic strength, etc.,which induces considerable changes in the native proteinstructure. Under such condition how it recognizes differ-ent drug/ligand molecules is a challenging field of investi-gation. Various binding and denaturation studies haveshown to be a rapid and effective tool for the characteriza-tion of albumin binding sites and their enantioselectivity,and for the study of the changes in the binding propertiesof the protein arising by interaction between differentligands. Here, we have reviewed two important aspects ofserum albumin, binding and various factors affecting con-

*Correspondence to: Rizwan Hasan Khan, Interdisciplinary BiotechnologyUnit, Aligarh Muslim University, Aligarh, India.E-mail: [email protected], [email protected]

Contract grant sponsors: Council of Scientific and Industrial Research,Department of Biotechnology, Department of Science and Technology.

Received for publication 29 July 2008; Accepted 19 January 2009DOI: 10.1002/chir.20709Published online in Wiley InterScience(www.interscience.wiley.com).

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formation of HSA, and predicted the possible strategiesthat could be implemented to improve the cargo of HSA.

LIGAND BINDING

Serum albumin possesses a unique capability to bind,covalently or reversibly, a great number of various endoge-nous and exogenous compounds. Several different trans-port proteins exist in blood plasma but albumin only isable to bind a wide diversity of ligands reversibly withhigh affinity. As conformational adaptability of HSAextends well beyond the immediate vicinity of the bindingsite(s), cooperativity and allosteric modulation ariseamong binding sites; this makes HSA similar to a multi-meric protein. However, it is important to address thisissue to obtain a fuller description of the ligand-bindingproperties. Albumin has a strong negative charge, butbinds weakly and reversibly to both cations and anions. Ittherefore functions as a circulating depot and transportsmolecules for a large number of metabolites including FA,ions, thyroxine, bilirubin and amino acids. More recentcrystallographic studies of a variety of drugs and biomole-cules complexes with HSA revealed the number and loca-tion of their binding sites on the protein. To date, the bind-ing sites for FA,2,8–11 hemin,12–14 thyroxine15 and a widearray of drugs16,17 have been identified. In recent yearshigh-resolution structural studies, coupled with the devel-opment of recombinant methods for expressing HSA inyeast, have stimulated renewed interest in the protein. Adetailed characterization of its binding properties is neces-sary not only for understanding its key physiological func-tions but also to help control its impact on drug deliveryand, most recently to facilitate development as a designeddelivery vehicle for a range of therapeutic and diagnosticreagents. In particular, studies with albumin mutants andcrystallographic studies of albumin-ligand complexes havegiven much new information about the location and struc-ture of binding sites and about potential ligand interac-tions. Figure 1 shows the primary ligands, binding onHSA which are highly adaptable further divided into dis-tinct subcompartments. These reveal a range of specificbinding sites distributed widely across the protein (Inset:highlighting the binding geometry of prototypical ligands).

FATTY ACID BINDING

Binding of a nonbinding compound to albumin can beachieved by coupling the compound to a ligand, such as afatty acid. High-resolution crystal structures of Fatty acid/HSA complexes revealed a total of seven binding sites dis-tributed heterogeneously throughout the protein andshared by medium-chain FA and saturated, monounsatu-rated or polyunsaturated long-chain FA.16,17 The bindingsites in HSA are compact and most specifically designedfor FA. The problem of deciphering the affinities of differ-ent binding sites is most acute for ligands such as Fattyacid, which bind to several different sites on the protein(see Fig. 1). In our previous work, we analyzed the effectsof fatty acid on binding of ligands and correlated the effect

of ligand binding on the unfolding of HSA.19 Single bind-ing of ligands to serum albumin is usually described ashigh-affinity binding to one or two sites and weaker bind-ing to a larger number of sites. It is important to note thatthe numbering of these sites in the crystal structure (fattyacid sites 1–7) was arbitrary and not based on affinity forfatty acid. Although the binding pockets appear welladapted to accommodate fatty acid, most of the bindingsites are capable of binding other ligand molecules. In par-ticular, drug site I (subdomain IIA) overlaps with fatty acidsite 7 and shares at least two amino acid side-chains withfatty acid site 2; and drug site II (subdomain IIIA) overlapswith fatty acid sites 3 and 4. Elsewhere on the protein,fatty acid site 1 also acts as the primary site for binding he-min, while fatty acid site 5 is a secondary site for propofoland thyroxine. Fatty acid site 6 is a secondary site for ibu-profen and diflunisal. Despite the repeating domain struc-ture of the protein, the fatty acid binding sites display asurprisingly asymmetric distribution. A large number ofarticles have been written on the nature and location ofthese binding sites and, although a definitive consensushas not been reached. Here (Figs. 2A–C), we have focusedon the interactions of the protein with its primary class ofligand and how HSA binds a variety of fatty acid mole-cules. The structures of the three HSA-fatty acid com-plexes [namely myristic complexed with thyroxine; C14(PDB ID, 1HK4); Stearic; C16 (PDB ID, 1E7I) and palmiticacids, C18 (PDB ID, 1E7H)] permit an extensive andhighly detailed survey of acids with the protein. Figure 2Ashows allosteric effect of thyroxine binding to HSA-myris-tic complex only at domain IIA. Using crystallographicanalyses four binding sites for thyroxine on HSA havebeen distributed in subdomain IIA, IIIA and IIIB.15 Thestructural observations depicts that HSA retains a high-af-finity site for thyroxine in the presence of excess fatty acidat IIA (drug site I) and IIIA (drug site II) mainly. The num-ber and location of binding sites for thyroxine are not welldefined although a consensus of most recent studies indi-cates that there are likely to be high affinity binding sitefor the thyroxine binding to serum albumin. For a morecomplete understanding of thyroxine binding to HSA invivo conditions where FA are invariably present, the inter-play between these two types of ligand needs to be charac-terized. Figure 2B shows the binding of medium chainfatty acid i.e. stearic acid (C18) to HSA. Both of these com-plexes in one way covers I-A, I-B and II-A while in otherway covers II-A, II-B, and III-A subdomains. Figure 2Cincludes one of the most physiologically important fattyacid ligands known to bind to HSA in vivo: palmitic acid(C16). Overall, we can conclude that there exists five toseven principal binding sites for FA ranging in chainlength from 14 to 18 carbon atoms.10–12

DRUG BINDING

Binding to HSA controls the free, active concentrationof a drug, provides a reservoir for a long duration of action,and ultimately affects drug absorption, metabolism, distri-bution, and excretion. The free concentration of a drug

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can also be affected by interaction with coadministereddrugs or by pathological conditions that can modify to asignificant extent the binding properties of the carrier,resulting in important clinical impacts for drugs that havea relatively narrow therapeutic index. The clinical conse-quences of drug-albumin interactions are now well under-stood. Dosage schedules that have been empiricallydevised for highly albumin-bound drugs are based on nor-mal concentrations and drug-binding behavior of albumin.HSA has a limited number of binding sites for endoge-nous22–33 and exogenous26,34–44 ligands (Table 1 and 2),so that drug binding to the protein may be affected by avariety of factors. The effect on pharmacokinetics of drug-drug competition for the same sites on HSA are generallyheld to be of little clinical importance.18,45 Physiological or

diseased states that cause variations in the plasma levelsof albumin or its primary endogenous ligands can influ-ence drug binding and may require dosages to be closelymonitored. Added to this, genetic polymorphisms in HSAcan also alter drug binding and may further complicatethe clinical picture.46,47 To understand the molecular basisof these effects, structural information is required to fullydelineate the binding sites for drugs and endogenousligands. Such information will also be invaluable to effortsto exploit the carrier properties of HSA in the developmentof novel therapeutic reagents for drug targeting48 or oxy-gen transport.49

Structural studies have mapped the locations of the fattyacid binding sites50,51 and the primary drug binding siteson the protein36 (Fig. 1). The fatty acid binding sites are

Fig. 1. Summary of the ligand binding capacity of HSA as defined by crystallographic studies to date. The domains are color coded as follows: I, ma-genta; II, green; III, blue. The A and B subdomains are depicted in light and dark shades, respectively. Inset shows binding location of prototypicalligands (bilirubin, PDB ID, 2VUE; thyroxine, PDB ID, 1HKI; diazepam, PDB ID, 2BXF and propofol, PDB ID, 1E7A) on respective domains.18

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distributed throughout the protein and involve all six sub-domains; by contrast many drugs bind to one of the twoprimary binding sites on the protein, known as Sudlow’ssites I and II.36,52 Although examples of drugs binding else-where on the protein have been documented,2,9,10 mostwork has focused on the primary drug sites. These investi-gations have largely employed competitive bindingmethods to investigate the selectivity of the primary drugbinding sites. Drug site I, where warfarin binds, has beencharacterized as a conformationally adaptable region withup to three sub domains. In addition, numerous subse-quent studies have shown that the presence of FA hasunpredictable effects on drug binding, and both coopera-tive and competitive interactions have been observed. Inmost cases, the mechanisms of interaction of these ligandswith HSA and the relative importance of the differentdomains and subdomains of HSA for ligand binding remainincompletely clarified. Therefore, the existence of the sitesdoes not, in itself, provide a complete explanation for theunique and complex ligand-binding properties of HSA.

Fig. 2. Crystal Structures of HSA complexed with three different fatty acids. Bound fatty acids are shown in yellow color space filling representation.This color scheme is maintained throughout. (A) myristic acid complexed with thyroxine (blue color) [PDB ID, 1HK4]15; (B) stearic acid [PDB ID, 1E7I]9;(C) palmitic acid [PDB ID, 1E7H]9; (D) the protein secondary structure is shown schematically with the domains color-coded as follows: I, magenta; II,green; III, blue. The dashed line shows GA module binding site at domain IIA.20,21 All Figures were prepared using Pymol software (version 0.99).

TABLE 1. Some groups of endogenous substances thatbind to albuminsa

CompoundAssociation

constant, K [M21] n Reference

Copper (II) 1.5 3 1016 1 22Hematin 1.1 3 108 1 23Long-chain fatty acids (1–69) 3 107 1 24Zinc (II) 3.4 3 107 1 22Bilirubin 9.5 3 107 25Thyroxine 1.6 3 106 1 26Eicosanoids 7 3 104 2 27Tryptophan 1.0 3 104 1 28Vitamin D3 5 3 104 1 29Bile Acids (3–200) 3 103 3 30Steroids (3.2–5) 3 103 1 –Calcium 6.5 3 102 3 31Magnesium 1 3 102 12 32Chloride 7.2 3 102 1 33

aAdapted from Peters.1

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STEREOSELECTIVE BINDING OF DRUGS

Stereoselective plasma protein binding of chiral drugmolecules administered in racemic form results in unevenenantiomeric composition of the unbound molecules.Besides quantitative differences the enantiomers may takepart in different kinds of binding interactions during simul-taneous binding with other drugs or endogenous ligands.In this situation, the single enantiomers act as distinctcompounds, their metabolism, distribution, and elimina-tion differing in principle. This accounts for the growinginterest in the molecular mechanisms involved in stereodiscrimination by serum proteins. Because of its ratherexceptional enantioselectivity, comparable to that dis-played by a specific drug target, HSA has also beenregarded as a ‘‘silent receptor’’. The different pharmacoki-netics profile of the drug bimoclomol enantiomers foundin humans is in accordance with the stereoselective highaffinity, and low capacity plasma protein binding.53 Fitoset al.54 found that, binding of (S)-warfarin involves mainlyacetonyl group located at the basic mouth of the bindingpocket where polar interactions occurs. The multiple effectof ibuprofen on lorazepam hemisuccinate showed competi-tive displacement for the common primary site in whichthe ionic interaction plays vital role, accompanied byinduced binding of the benzodiazepine due to allostericmodification of the protein conformation.55

BACTERIAL PROTEIN AND ALBUMIN ASSOCIATION

In the complex molecular interplay between a pathogenand its human host, bacterial binding association plays im-portant roles. GA (protein G-like albumin binding module)is found in a family of surface proteins of different bacterial

species. It has been reported that the GA module bindsclose to a cleft at a site in domain IIA and domain IIIB ofthe albumin molecule20,21 and involves a single site con-sisting of a segment spanning residues 330–548 (Fig. 2D).This module binding to HSA might prove useful, espe-cially in the context of minimizing the HSA affinity of drugmolecules by structure-based drug design.

METAL BINDING

Acute and chronic toxicoses caused by heavy metal ionsmainly involving cadmium, mercury and lead are knownfor all forms of life.56,57 Moreover, cadmium and verylikely lead are carcinogens in humans,58,59 and may alsoinduce neurodegenerative diseases.60,61 HSA is useful as amodel system for studying the metal binding properties ofother vertebrate serum proteins. A large effort has beendevoted to the study of the metal-binding sites of albumin,but a detailed discussion of this particular binding prop-erty of the albumins is beyond the scope of this review. Itsuffices to say that N terminal portion of HSA (N-Asp-Ala-His-Lys-) possess high affinity for Cu(II), Ni(II), Hg(II),Au(I), and Ag(II) with weaker affinities for Ca(II) andZn(II). Of particular importance for the binding of metalsis Cys34 (site V) and the N terminus of the protein (siteVI). Copper (II) and nickel (II) deserve special considera-tion (region 4) among the metals because most mamma-lian albumins bind them more tightly and more specifi-cally than they do other cations. HSA has also beenreported to possess a relatively weak, nonspecific; latentiron-binding capacity.62 This is, however, unlikely to be ofsignificance under normal circumstances in plasma,because the specific, high affinity, iron binding proteintransferrin binds all low-molecular-mass ferric ions.

ALLOSTERIC INTERACTIONS

Allosteric effects occur when the interaction of one sub-stance with a binding agent alters the interactions of a sec-ond substance at a different region on the same agent.Such effects can occur during target-receptor and drug-protein binding.55,63 The globular domain structural orga-nization of monomeric HSA is at the root of its allostericproperties which are reminiscent of those of multimericproteins. Information about competitive and synergisticinfluences of ligands is important, because alteration inprotein binding may alter the volume of distribution, clear-ance, and elimination of a drug and may modulate its ther-apeutic effect.

Drug-Drug Interactions

Allosteric interactions have been observed between anumbers of drugs as they bind to HSA. Drug-drug interac-tions at the protein-binding level are usually regarded asproblematic secondary effects but useful for therapeuticpurposes.64 Binding of drugs to plasma proteins is an im-portant determinant for their biological efficacy since itmodulates drug availability to the intended target. Benzo-diazepines bind to several HSA clefts depending on their

TABLE 2. Binding of drugs and other exogenouscompoundsa

CompoundAssociation

Constant, K [M21] n Reference

Site I (Subdomain II A)Indomethacin 1.4 3 106 1 34Bromphenol blue 1.5 3 106 3 35Salicylate 1.9 3 105 1 36Warfarin 3.3 3 105 1 37Phenylbutazone 7.0 3 105 1 36Digitoxin 0.4 3 105 1 26Furosenamide 2.6 3 104 1.6 38Phenytoin 6 3 103 6 39Chlorpropamide 3.3 3 105 1 36Benzylpenicillin 1.2 3 103 1 40Evans blue 4.0 3 105 14 41

Site II (Subdomain III A)Diazepam 3.8 3 105 1 42Ibuprofen 2.7 3 106 1 26Naproxen 1.2 3 106 1 43Clofibrate 7.6 3 105 1 44Chlorpromazine 2.0 3 105 – 26Imipramine 2.5 3 104 – 26Quinidine 1.6 3 103 – 26

aAdapted from Peters.1

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conformation and substitution. The most remarkable allo-steric effect could be observed in the simultaneous bind-ing of (S)-lorazepam acetate65 clonazepam and (S)-uxe-pam.66 These drugs selectively increase the binding of (S)-warfarin to HSA.

Drug-Non drug Interactions

At higher relative concentrations, the medium chainfatty acid anions often displace site II drugs by competitionwhile the displacing effect in site I drugs is probably verysmall. The improving effect of long-chain fatty acid anionson the binding of certain site I ligands was originallyexplained by induction of conformational changes in the al-bumin molecule rendering site I more suitable for ligandbinding. For example consider binding of 2,3,5-triiodoben-zoate (TIB) at sites I and II in absence of myristic acid.2 Inthe presence of myristate, TIB becomes displaced fromsite II but rebinds at a new, myristate-induced binding sitein subdomain IB.11 Remarkably, Arg218-His and Arg218-Pro mutations within subdomain IIA greatly enhance theaffinity for thyroxine and causes the elevated serum totalthyroxine levels associated with familial dysalbuminemichyperthyroxinemia.15 Myristate not only competitivelybinds to FA1, but allosterically modulates the affinity ofthe Mn (III) heme label to FA1 in a complex way; interest-ingly, the myristate affinity for the modulatory site is allo-sterically modulated by a third FA binding site.67 The fer-ric heme allosterically regulates inhibition of drug bindingto HSA sudlow’s site I. In turn, drugs impair allostericallyheme binding to HSA by the same extent, in agreementwith spectroscopically and conformationally linked func-tions.68 The increase in plasmatic levels of ferric hemeunder pathological conditions (e.g. hemolytic anemia) mayinduce a massive release of drugs with concomitant intoxi-cation of the patient. Newborns are suggested to be testedagainst bilirubin-displacing properties since strong displac-ing drugs such as valproate, sulfisoxazole, phenylbuta-zone, glibenclamide, tolbutamide, and furosemide shouldbe avoided under high conditions of kernicterus.68

HSA is widely used as a model multidomain protein toinvestigate how interdomain interactions affect the globalfolding/unfolding process. The binding affinity of HSA ishighly dependent on its conformational changes. Undersuch conditions how it recognizes different drug/ligandmolecules is a challenging field of investigation.

CONFORMATIONAL CHANGES AFFECTINGLIGAND BINDING

The structure and dynamics of HSA are known to beinfluenced by different environmental conditions, whichon the other hand depend on several factors like, tempera-ture,69 pH,70 ionic strength,32 surfactants,71,72 and chemi-cal denaturants73–78 yielding basic information about possi-ble hydrophobic and electrostatic interactions, contribut-ing to the overall binding affinity. In several disease states,such as bacterial infection, diabetes mellitus, and stress,the fatty acid levels are found to increase remarkably.Under such circumstances, the fatty acid modulates the

ligand binding properties of HSA by inducing conforma-tional changes in the binding sites I and II.19

A schematic model represented the spatial relationshipsamong drug site I, Trp-214, and Cys-34 along with the pal-mitic acid as probes. The instantaneous loss in the bindingproperties of environmental sensitive probe, prodan ondenaturation supports the idea that the unfolding of HSAoccurs with an initial separation of domain I and domain IIresulting in the expanded form of the protein followed bycomplete unfolding of the domains.79 HSA acts as an effec-tive plasma buffer due to the presence of many chargedresidues. At physiological pH, albumin has a net charge ofnegative 19. It is responsible for about half of the normalanion gap. A reduction in plasma protein concentrationcauses metabolic alkalosis.80 HSA is known to exist asNeutral ‘N’ (pH�7) and basic ‘B’ isomeric forms underequilibrium conditions. This N-B transition has physiologi-cal significance and is suggested by the fact that, underincreased Ca21 ions concentration in blood plasma, the Bisomer predominates. Moreover, it is believed that thetransport function of albumin is controlled through thistransition or akin to it.70 The N$B isomerization is inter-preted to be ‘‘a structural fluctuation, a loosening of themolecule with higher configurational adaptability’’. Warfa-rin and ibuprofen binding stabilizes heme-HSA in both Nand B states.81 Complexation of HSA with amphiphilic anti-depressant drug amitriptyline82 and penicillin83 at pHabove, below and isoelectric point of the protein, evi-denced saturation below CMC (critical micellar concentra-tion) and conformational change above CMC. Pedersenet al.84,85 showed that the binding strength of the FA doesnot change much in the acid to neutral pH region, butbecomes stronger when moving towards alkaline region.The recent advancement in the field of nanosciencerequires the preparation of bioactive nanoparticles underdifferent temperatures using protein molecules as tem-plates.86–89 The single-residue mutations could modify thestability of albumin and that the effects are more pro-nounced for mutations in sub domain IIA (sudlow site I)than for mutations in sub domain IIIA (sudlow site II).Similar approach was being made to characterize the si-multaneous binding of the ligands in various temperature-dependent folded states of HSA for revealing the nature ofbinding of ligands. A portion of the cationic probe NB,which resides in subdomain IIB at room temperature, wasexpelled from its original location at higher temperaturesdue to melting of subdomain at elevated temperatures.90

Reversible thermal dentauration of genetic variants of HSAespecially in domains I and III provides more insights intoboth protein chemical relevance and of clinical interest,because they could be useful when designing stable,recombinant HSA for clinical application.91

PRACTICAL ASPECTS OF LIGANDBINDING STRATEGIES

Medically, a generous concentration of albumin in theblood stream is a measure of the ‘‘Quality of Life.’’92 Bind-ing of exogenous/endogenous ligands and unfolding/

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refolding of HSA appears to be relevant in drug therapyand management. The usual paradigm is that ligand bind-ing induces a change in the conformation of the target pro-tein that, in turn, produces a given response. In one hand,albumin can carry almost all chemical entity present in theblood including drugs, and in the other hand its structurecan be engineered to different extents with the help ofcheap and easily available chemical entities. It is wellknown that protein stability is modified by ligand bindingdue to the coupling between two mutual processes underequilibrium: binding and unfolding.93–95 Some improve-ments in existing methodologies, new applications ofexperimental techniques, and development of new ap-proaches aimed have presented here:

1. HSA can be used to clear the body of endogenous tox-ins. For example we can design an albumin basedextracorporeal detoxifier (the previous known one isMARS; molecular adsorbent recirculating system).96

Technically speaking, depending on the binding affin-ity with endogenous ligand, an albumin-containing di-alysis that can recirculate and regenerate online by di-alysis against buffered solutions, followed by passagethrough adsorbent (like charcoal) and anion-exchanger columns. This method of removing albu-min-binding toxins and water-soluble substanceswould result in hepatic-encephalopathy and inimprovements in kidney and liver function.97,98

2. HSA is widely distributed in the plasma compartmentand easily related to the immune system. The forma-tion of acyl glucuronides is a major metabolic pathwayfor many compounds with a carboxylic acid function.This type of metabolite is a reactive electrophilic spe-cies and can therefore, in addition to reversible bind-ing react covalently with HSA both in vitro and invivo. Several drugs become glucuronidated and inter-act covalently with HSA, include mainly NSAIDs liketolmetin99 and furosemide.100 Binding of these drugsmay predict the degree to which the correspondingacyl glucuronides form covalent adducts that prob-ably/possibly leads to toxicity. This information couldbe a useful adjunct in drug design.

3. Enantiomeric forms of a drug can differ in potency,toxicity, and behavior in biological systems. There-fore, many chiral analytical and preparative methodshave been developed for the separation of drug enan-tiomers.101–106 Immobilized albumins have used aschiral selector to separate enantiomers of trypto-phan,107,108 several organic compounds107,108 includ-ing drugs (oxazepam,109 arylcarboxylic acids110 andwarfarin108) ethacrynic acid111,112 and neoglycopro-teins.113 The ability of immobilized albumin to sepa-rate drug enantiomers has also been used in affinitycapillary electrophoresis to separate racemic drugs.114

4. Apart from its vital role in transporting drugs and en-dogenous compounds, HSA is also involved in theinactivation of a small group of compounds resultingin enzymatic activity. HSA acts like thioestearsebecause it has a free sulfhydryl group at cys.37 Forexample, the protein can degrade drug disulfiram

(Antabuse), a clinically important process.115 HSA cancatalyze dehydration of prostaglandin D2

116 and E2.117

Members of the penem group of antibiotics binds irre-versibly to albumin through acetylation of an e-lysineto amino acid residues of site I, with the R-isomerbeing degraded much faster that the S-isomer.118 Peni-cillin allergy has been linked to irreversible couplingof penicilloyl groups to these lysine groups.119 Theresulting complex may be clinically significant.

5. HSA accounted for about 40% of the total plasma activ-ity, and the esterase like domain was assigned to sub-domain IIIA of which Tyr411 should be important. Thisdomain on isolation can be used to detoxify cyanidesby forming binding conjugates of albumin with sulfurforming thiocyanates,120 p-nitrophenyl acetate119,121

and several N-carbobenzoxy-DS (L)-alanine-p-nitro-phenyl esterase.122 A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), was found to bemore reactive than the classical o-nitroacetanilide,thereby possessing aryl acylamidase activity of fatty-acid free HSA.123 From the pharmaceutical and clini-cal points of view it is interesting that the esterase likeactivity of HSA can be used to activate prodrugs suchas olmesartan medoxomil to the active drug olmesar-tan.124 However, this kind of prodrug activation can beclinically relevant, because although the enzymatic ac-tivity of a single HSA molecule is low, the concentra-tion of the protein in the circulation is very high.

6. Free HSA could be used as a useful noncovalentlybound vehicle for the highly insoluble and toxic zincphthalocyanine, a second-generation photo sensitizerfor the photodynamic therapy of cancer.125 This couldprovide a simpler alternative for liposome incorpo-rated zinc phthalocyanine which in turn appeared toeffectively control tumor growth in a human colon car-cinoma, T380.

7. Since chemotherapeutics agents are notorious fortheir side effects. HSA is a marvelous binder of thera-peutical drugs. For effective therapy of disseminatedmalignant diseases, it can be used as a shielding mem-brane around target organs so that disease can notspread and side effects can be minimized.

8. The big advantage of albumin is the compatibility withhuman blood, plasma, and body components. Thus,HSA can be used as a cargo for immobilization of hor-mones covalently coupled to the endogenous ligandsin vivo such as FA. A strategy can be designed to opti-mize the pharmacokinetics of insulin and especiallythe glucose disposal curve elicited by insulin. The hor-mone was covalently coupled to FA, which bind to se-rum albumin in vivo. This appeared to result in favor-able pharmacokinetics but especially in improved glu-cose disposal curve.125–129

9. With the advancement of Bioinformatics, the survey ofthe albumin binding characteristics of major pharma-ceutical compounds could lead to the discovery of pre-viously unidentified drug binding sites and possiblebinding mechanisms, creating major opportunities fordesigning safer and more effective drugs. This sort ofresearch required the cloning and expression of several

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species of albumins, albumin fragments and albuminmutants as well as antibodies to these molecules.

10. Furthermore, successful production of recombinantHSA (rHSA) by secretion from yeast cells77 both in S.cerevisiae130 and K. lactis131 with no apparent loss ofbinding properties could be utilized for further muta-genesis studies to check the role and importance ofthe residues proposed above in the binding process.

11. Successful designing of the HSA micro spheres couldbe used for the delivery of cytostatic agents such asdoxorubicin and 5-fluorouracil to tumors in the liver,breast, and lungs, rendering the albumin-bound drugsmore effective than free drug.132 Albumin-propyleneglycol alginate-coated covalent network offers a betterresistance to the microspheres towards freezing, ly-ophilization and sterilization.133 A composite collagenhydrogel containing protein encapsulated alginatemicrospheres was also developed for ocular applica-tions.134

ACKNOWLEDGMENTS

Facilities provided by A.M.U are gratefully acknowl-edged. The authors are also thankful to DST (FIST) forproviding lab facilities.

LITERATURE CITED

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